Throughout this application various publications are referenced within parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains.
1. The Field of the Invention
This invention relates to the medical arts. In particular, it relates to the field of genetic testing methods and diagnostic kits.
2. Discussion of the Related Art
Statin drugs--the most potent lipid-lowering agents currently available--are 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors. They include lovastatin, pravastatin, simvastatin, atorvastatin, fluvastatin, and cerivastatin. All these statin drugs share a common mechanism of action and have similar toxicity profiles. (E. von Kreutz and G. Schluter, Preclinical safety evaluation of cerivastatin, a novel HMG-CoA reductase inhibitor, Am. J. Cardiol. 82(4B):11J-17J [1998]; A. G. Ollson [1998]).
The statin drugs are effective in reducing the primary and secondary risk of coronary artery disease and coronary events, such as heart attack, in middle-aged and older men and women (under 76 years), in both diabetic and non-diabetic patients, and are often prescribed for patients with hyperlipidemia. (A. G. Ollson, Addressing the challenge, Eur. Heart J. Suppl. M:M29-35 [1998]; M. Kornitzer, Primary and secondary prevention of coronary artery disease: a follow-up on clinical controlled trials, Curr. Opin. Lipidol. 9(6):557-64 [1998]; M. Farnier and J. Davignon, Current and future treatment of hyperlipidemia: the role of statins, Am. J. Cardiol. 82(4B):3J-10J[1998]). Statins used in secondary prevention of coronary artery or heart disease significantly reduce the risk of stroke, total mortality and morbidity and attacks of myocardial ischemia; the use of statins is also associated with improvements in endothelial and fibrinolytic functions and decreased platelet thrombus formation. (M. Kornitzer [1998]; M. Farnier and J. Davignon, Current and future treatment of hyperlipidemia: the role of statins, Am. J. Cardiol. 82(4B):3J-10J [1998]).
The use of statin drugs has recently decreased the need for surgical coronary revascularization, known as coronary artery bypass graft (CABG). (B. M. Rifkind, Clinical trials of reducing low-density lipoprotein concentrations. Endocrinol. Metab. Clin. North Am. 27(3):585-95, viii-ix [1998]). But CABG is still a common surgical intervention for patients who develop atherosclerotic occlusion in coronary arteries. Approximately 12,000-14,000 CABG procedures are performed annually. (G. F. Neitzel et al., Atherosclerosis in Aortocoronary Bypass Grafts, Atherosclerosis 6(6):594-600 [1986]). The patient's own saphenous vein, or brachial or mammary artery, is used to bypass the affected coronary artery. The majority of CABG patients experience good long-term results, but 30-40% require a second CABG within 10-12 years after surgery, and continuing atherosclerosis in the graft is an important factor in late graft failure. (L. Campeau et al., The effect of aggressive lowering of low-density lipoprotein cholesterol levels and low-dose anticoagulation on obstructive changes in saphenous-vein coronary-artery bypass grafts, N. Eng. J. Med. 336(3):153-62 [1997]).
Atherosclerosis in bypass grafts is associated with elevated serum levels of very low density lipoproteins (VLDL), low density lipoprotein cholesterol (LDL-C), and triglycerides, and low levels of high density lipoprotein cholesterol (HDL-C). (J. T. Lie et al., Aortocoronary bypass saphenous vein atherosclerosis: Anatomic study of 99 vein grafts from normal and hyperlipoproteinemic patients up to 75 months postoperatively, Am. J. Cardiol. 40:906 [1977]; L. Campeau et al, The relation of risk factors to the development of atherosclerosis in saphenous vein bypass grafts and the progression of disease in the native circulation, N. Eng. J. Med. 311(21): 1329-32 [1984]). It is standard for CABG patients to be prescribed statin drugs to lower their serum LDL-C.
Lipid lowering therapy has been demonstrated to delay the progression of atherosclerosis in coronary arteries. (E.g., G. Brown et al., Regression of coronary artery disease as a result of intensive lipid lowering therapy in men with high levels of apolipoprotein B, N. Engl. J. Med. 323:1289-98 [1990]; J. P. Kane et al., Regression of coronary atherosclerosis during treatment of familial hypercholesterolemia with combined drug regimens, JAMA 264:3007-12 [1990]; Jukema et al., 1995). Prior to the Post-CABG Trial, few data were available to determine the efficacy of LDL-lowering therapy to delay the obstruction of saphenous-vein grafts. (D. H. Blankenhorn et al., Beneficial effects of combined colestipol-niacin therapy on coronary atherosclerosis and coronary venous bypass grafts, JAMA 257:3233-40 [1987]). Furthermore, thrombosis had also been observed to contribute to graft obstruction (G. F. Neitzel et al., Atherosclerosis in aortocoronary bypass grafts. morphologic study and risk factor analysis 6 to 12 years after surgery, Arteriosclerosis 6:594-600 [1986]). Low-dose anticoagulation therapy prevented emboli after major surgery (A. G. G. Turpie et al., Randomised comparison of two intensities of oral anticoagulant therapy after tissue heart valve replacement, Lancet 1:1242-45 [1988]; L. Poller et al., Fixed minidose warfarin: a new approach to prophylaxis against venous thrombosis after major surgery, Br. Med. J. 295:1309-12 [1987]), and this implied that low-dose anticoagulation treatment would also be able to delay graft obstruction.
Statin drug treatment beneficially affects the long-term outcome for most CABG patients. In a large clinical study, the Post-CABG Trial, CABG patients received statin drug treatment to lower serum LDL-C; in comparing patients who had received aggressive lovastatin treatment (LDL-C lowered to 93-97 mg/dl) to those who had only received moderate lovastatin treatment (LDL-C lowered to 132-136 mg/dl), the percentages of patients with atherosclerotic worsening of grafts within 4 years were 39% and 51%, respectively,. (L. Campeau et al. [1997]). The number of patients in the aggressive lovastatin-treatment group who required a second CABG procedure was 29% lower than the number in the moderate-treatment group.
In addition to serum lipid concentrations, there are other risk factors, that may have a genetic basis, and that may independently affect atherosclerotic coronary artery disease and occlusion of bypass grafts or that interact with statin treatment to lower serum lipids, which can affect atherosclerotic stenosis. Several laboratories have observed a link between variant alleles of the lipoprotein lipase gene (LPL) and the occurrence and/or progression of atherosclerosis. The involvement of LPL in coronary artery disease was suspected, since rare homozygotes for defects in this gene have type I hyperlipoproteinemia (OMIM 238600) and premature coronary artery disease. (P. Benlian et al., Premature atherosclerosis in patients with familial chylomicronemia caused by mutations in the lipoprotein lipase gene. N. Engl. J. Med. 335:848-54 [1996]).
Lipoprotein lipase (LPL; E.C. 3.1.1.34), also known as triacylglycerol acylhydrolase, is a heparin-releasable glycoprotein enzyme bound to glycosaminoglycan components of macrophages and to the luminal surface of capillary epithelial cells in a variety of human tissues, including heart, skeletal muscle, adipose, lung, and brain. (K. L. Wion et al., Human lipoprotein lipase complementary DNA sequence, Science 235:1638 [1987]; C. Heizmann et al., DNA polymorphism haplotypes of the human lipoprotein lipase gene: possible association with high density lipoprotein levels, Hum. Genet. 86:578-84 [1991]). Lipoprotein lipase is active as a dimer of identical subunits, each approximately 62,500 D in unglycosylated form. (M. R. Taskinen et al., Enzymes involved in triglyceride hydrolysis. In: James Shepard (Ed.), Bailliere's Clinical Endocrinology and Metabolism, Vol. 1, No.3, Bailliere Tindall, London, pp.639-66 [1987]).
Lipoprotein lipase is the rate-limiting enzyme for the hydrolysis and removal of triglyceride-rich lipoproteins, such as chylomicrons, VLDL, and LDL-C from the blood stream. (Jukema et al., The Asp.sub.9 Asn Mutation in the Lipoprotein Lipase Gene Is Associated With Increased Progression of Coronary Atherosclerosis, Circulation 94(8):1913-18 [1996]). The enzymatic action of LPL results in the generation of mono- and diglycerides and free fatty acids that can be used as fuel for energy or reesterified for storage in peripheral adipose tissue.
The gene sequence of human LPL is known, including the 3' region through exon 10 and the 3' untranslated region (3'-UTR). (K. L. Wion et al., Human lipoprotein lipase complementary DNA sequence, Science 235:1638-41 [1987]; T. G. Kirchgessner et al., The sequence of cDNA encoding lipoprotein lipase, J. Biol. Chem. 262(18):8463-66 [1987]; K. Oka et al., Structure and polymorphic map of human lipoprotein lipase gene. Biochim. Biophys. Acta 1049:21-26 [1990]; D. A. Nickerson et al., DNA sequence diversity in a 9.7-kb region of the human lipoprotein lipase gene, Nat. Genet. 19:233-40 [1998]). Nickerson et al. sequenced the region of the LPL gene spanning exons 4-9 (containing the major catalytic portion of the enzyme) of 71 individuals taken from 3 different populations and observed 88 different DNA variants or polymorphisms, with 78 of these present at an allele frequency greater than 1% (D. A. Nickerson et al., [1998]).
Two LPL polymorphisms are known to affect LPL activity. The D9N mutation in exon 2 has been associated with increased triglyceride levels and with the occurrence of coronary atherosclerosis, attenuating the ability of pravastatin to lower LDL-C. (J. Jukema et al. [1996]). The N291S mutation in exon 6 has been associated with reduced HDL-C levels. (P. Reymer et al., A lipoprotein lipase mutation [asn291ser] is associated with reduced HDL cholesterol levels in premature atherosclerosis, Nat. Gen. 10:28-34 [1995]; H. H. Wittrup et al., A common substitution [asn291ser] in lipoprotein lipase is associated with increased risk of ischemic heart disease, J. Clin. Inves. 99:1606-13 [1997]). The N291S mutation is also linked with increased coronary stenosis (narrowing of arterial lumen) seen on angiography in women with verified ischemic heart disease compared to controls. (H. H. Wittrup et al. [1997]).
Two other LPL polymorphisms have demonstrated association with the development of atherosclerosis, although their functional significance is unknown. The first is the PvuII polymorphism in intron 6, which is linked with the number of coronary blood vessels with greater than 50% obstruction. (X. Wang et al., Common DNA polymorphisms at the lipoprotein lipase gene: association with severity of coronary artery disease and diabetes, Circulation 93:1339-45 [1996]). The second is the HindIII polymorphism in intron 8, associated with the angiographic severity of coronary artery disease. (R. Mattu et al., DNA variants at the LPL gene locus associate with angiographically defined severity of atherosclerosis and serum lipoprotein levels in a Welsh population, Arterio. Thromb. 14:1090-97 [1994]; R. Peacock et al., Associations between lipoprotein lipase, lipoproteins and lipase activities in young myocardial infarction survivors and age-matched healthy individuals from Sweden, Atherosclerosis 97:171-85 [1992]).
Progress in pharmacogenetics has shown that human genetic variation underlies different individual responses to drug treatment within a population. (Reviewed in G. Alvan, Genetic polymorphisms in drug metabolism, J. Int. Med. 231:571-73 [1992]; P. W. Kleyn and E. S. Vesell, Genetic variation as a guide to drug development, Science 281:1820-22 [1998]). For example, alleles of the NAT1 and NAT2 genes (N-Acetyltransferases) create a "slow acetylator" phenotype in 40-60% of Caucasians, resulting in a slow clearance and associated toxicity of many drugs including isoniazid and procainamide (K. P. Vatsis et al., Diverse point mutations in the human gene for polymorphic N-acetyltransferase, Proc. Natl. Acad. Sci. USA 88(14):6333-37 [1991]). A defect in CYP2D6 (a member of the cytochrome P450 family) leads to the "poor metabolizer" phenotype in 5-10% of Caucasians, affecting the metabolism of many drugs including some beta-blockers and antiarrhythmics. (Reviewed in A. K. Daly et al., Metabolic polymorphisms, Pharmac. Ther. 57:129-60 [1993]). Some genetic variation can be associated with the accumulation of toxic products, for example treatment of TPMT-deficient (thiopurine methyltransferase) patients with 6-mercaptopurine or azathioprine can lead to a potentially fatal hematopoietic toxicity due to higher than normal levels of thioguanine nucleotides. (R. Weinshilboum, Methyltransferase pharmacogenetics, Pharmac. Ther. 43:77-90 [1989]; E. S. Vesell, Therapeutic lessons from pharmacogenetics, Ann. Intern. Med. 126:653-55 [1997]).
The presence of multiple genetic and environmental factors capable of creating such large variations in how drugs operate in the patient argues that individualization of the choice of drug and dosage is required for optimal treatment of disease, including atherosclerotic coronary artery disease. Jukema et al. (1996) reported that the HMG-CoA reductase inhibitor pravastatin did not lower the LDL-cholesterol level in subjects with the LPL N9 polymorphism to the same extent as in those with the LPL D9 polymorphism. In addition, J. A. Kuivenhoven et al. (1998) observed that pravastatin slowed the progression of atherosclerosis in subjects with the CETP B1B1 genotype, but not in those with the CETP B2B2 genotype. (J. A. Kuivenhoven et al., The role of a common variant of the cholesteryl ester transfer protein gene in the progression of coronary atherosclerosis, N. Engl. J. Med. 338:86-93 [1998]). These reports suggest that there are interactions between statin drugs and some genetic determinants of atherosclerosis.
There has been a definite need for a reliable predictive test for determining which patients suffering from coronary artery disease, or which CABG patients, will likely not respond positively to statin drug treatment with respect to stenosis of a coronary artery or bypass graft. Such a genetic testing method can provide useful information so that patients can be given more individually suited alternative treatments to prevent further injury.
This and other benefits of the present invention are described herein.